A new step towards understanding the mechanism of cell reprogramming
American scientists have taken another step towards unraveling the mechanism of the process of creating induced pluripotent stem cells. By combining mouse embryonic stem cells with human fibroblasts to create heterokaryons – cells containing two or more nuclei and having different genotypes – Helen Blau and colleagues from Stanford University have developed a new way to study the process of cellular reprogramming. Researchers believe that the suppression of fibroblasts by stem cell growth factors leads to more efficient and accelerated reprogramming of human nuclei in heterokaryons. Thus, in Blau's experiments, already on the second day after fusion, 70% of cells showed expression of pluripotency markers – a huge jump compared to less than 0.1% in the case of inducing pluripotency using all the techniques developed so far.
Using the new method, the researchers studied the role of DNA demethylation, one of the most important steps in the process of reprogramming fibroblasts into induced pluripotent stem cells. As a result, it was found that in reprogrammed heterokaryons, demethylation occurs on the promoters of the well-known pluripotency genes OCT4 and NANOG, and also correlates with an increase in the expression of both genes. In addition, scientists have observed demethylation in the absence of cell division and DNA replication, which, contrary to popular beliefs, allows us to make an assumption about the active nature of this process during reprogramming.
To understand the mechanism of demethylation, scientists focused their efforts on an enzyme called "activation-induced cytidine deaminase AID" (activation-induced cytidine deaminase AID), previously discovered in germ cells and presumably performing a specific function in the overall demethylation in the embryos of the danio fish. A day before the experiment, scientists blocked AID in mouse stem cells and human fibroblasts. In the resulting heterokaryons, the expression of both OCT4 and NANOG genes was significantly weaker, as was demethylation. Overexpression of AID in these cells completely excluded demethylation and expression of NANOG and partially OCT4. Thus, demethylation of two key pluripotency genes is an important stage of cellular reprogramming, in which the AID enzyme plays a decisive role.
Without stopping there, the researchers continued their experiments and confirmed AID's direct involvement in the demethylation process. The DNA demethylation reaction plays an important role in the development of mammals, its importance in the development of oncological diseases and the aging process has been noted, but the key factors of this process have not yet been known. Dr. Blau is in no hurry to unconditionally attribute demethylation to the AID enzyme and believes that it does not act alone. According to her, the exact mechanism of demethylation still seems rather vague. The authors of the study believe that AID somehow initiates the DNA repair process, replacing methylated bases with unmethylated ones, but its details have yet to be worked out.
The scientists plan to establish whether AID works similarly in other methods of inducing pluripotency, and also whether AID supplements can increase the effectiveness of these methods.
The authors also hope that the heterokaryon method will allow them to finally reveal the mechanism of cellular reprogramming, the first step to which they have already taken by identifying a key factor in the process of DNA demethylation.